Hepatitis C virus proliferation inhibitor

ABSTRACT

A hepatitis C virus proliferation inhibitor comprising a bile acid or a physiologically acceptable salt thereof is disclosed, as well as a method for treating a mammal infected with hepatitis C and a pharmaceutical composition for treating hepatitis C. As the bile acid to be used in the present invention, free bile acids, such as ursodeoxycholic acid and chenodeoxycholic acid, and conjugated bile acids such as tauroursodeoxycholic acid are exemplified. Also, as the physiologically acceptable salt, alkali metal salts such as the sodium salt of the bile acid is exemplified.

This application is a 371 of PCT/JP94/01187 filed Jul. 19, 1994,published as WO95/03056 Feb. 2, 1995.

FIELD OF THE INVENTION

The present invention relates to a hepatitis C virus proliferationinhibitor containing bile acid as the active principle.

BACKGROUND ART

Hepatitis, which is neither hepatitis A nor hepatitis B, forms 95 to100% of post-transfusion hepatitis and 40 to 50% of sporadic hepatitisand easily becomes chronic, further changing at high rates to cancer ofliver via chronic hepatitis or hepatic cirrhosis. Recently, hepatitis Cvirus (hereinafter referred to as HCV) was identified, and it has beendemonstrated that most of hepatitis previously known as non-hepatitis Aor non-hepatitis B are caused by this hepatitis C virus.

Although interferon has been known as an agent having inhibitory effecton the proliferation of hepatitis C virus, it is pointed out that thereare problems such as its low rate in the effectiveness as little as 30to 40%, the 60 to 70% recrudescence after discontinuance of the dosagethereof, the appearance of influenza-like symptoms, such as pyrexia,headache and vomiting, and of diverse side effects such as leukopenia,at the high rates.

Bile acid is a drug being publicly known as a choleretic, liver functionimproving agent, etc. As examples of the inhibitory effect of bile acidon the proliferation of virus, the anti-viral activities against herpesvirus, human immunodeficiency virus, influenza virus, parainfluenzavirus, etc., have been known (see Japanese Patent Laid-opened Nos. Sho59-167517 Gazette, Sho 63-301823 Gazette, Hei 2-167235 Gazette, Hei4-500514 Gazette and Hei 4-500670 Gazette). However, it has not beenknown that bile acid has an inhibitory effect on the proliferation ofhepatitis C virus. Although a suppository preparation comprising anursodeoxycholic acid salt and interferon is disclosed in Japanese PatentLaid-opened No. Sho 62-77333 Gazette, the bile acid is used just as anabsorption promoting agent in the dosage of interferon into colon orrectum, and therefore, the disclosure does not suggest at all that bileacid has an inhibitory effect on the proliferation of hepatitis C virus.

It is an object of the present invention to provide a novel and highlyeffective hepatitis C virus proliferation inhibitor which gives noadverse reaction.

DISCLOSURE OF THE INVENTION

As a consequence of enthusiastic investigation made by the inventors ofthe present invention on hepatitis C and hepatitis C virus, suchinventors found that bile acid can show an inhibitory effect on theproliferation of hepatitis C virus and have subsequently accomplishedthe present invention. Namely, the present invention is directed to ahepatitis C virus proliferation inhibitor comprising bile acid or thephysiologically acceptable salts thereof as the active principle.

As bile acid to be used in the present invention, free bile acid, suchas ursodeoxycholic acid and chenodeoxycholic acid, or conjugated bileacid such as tauroursodeoxycholic acid can be exemplified. As thephysiologically acceptable salt of bile acid, an alkali metal saltthereof such as the sodium salt can be exemplified. These salts can beprepared according to any of the methods for manufacturing bile acidwidely known in the art.

As the administration route for the hepatitis C virus proliferationinhibitor according to the present invention, oral administration withany of tablets, capsules, granules, powders, medicated syrup, etc. orparenteral administration with injections,. suppositories, etc. can beexemplified. These pharmaceutical preparations can be prepared bycombining with additives, such as fillers, binders, disintegrators,lubricants, stabilizers and correctives, and according to any of themethods widely known in the art. The dose range of the inhibitory agentfor an adult per day, though subject to symptoms and ages of thepatients as well as other factors, is normally from 800 to 3,000 mg,preferably from 1,200 to 1,800 mg, and more preferably from 1,400 to1,600 mg, for oral administration, and from 30 to 1,200 mg, preferablyfrom 50 to 1,000 mg, and more preferably from 100 to 600 mg, forparenteral administration, and the dosage can be made either at onetime, or divided into several times a day. In the case of parenteraladministration, it is preferable to dose the agent according to theintravenous drip infusion method.

BEST MODE FOR CARRYING OUT THE INVENTION

The results each obtained in inhibition tests on the proliferation ofhepatitis C virus, clinical tests and acute toxicological tests withbile acid are described hereinafter in detail. For the bile acid,ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA) wereused as the representative compound.

(Inhibition Test on Proliferation of Hepatitis C Virus)

Liver biopsy was carried out on six patients aged from 40 to 60 who aresuffering chronic hepatitis C and positive to both HCV antibody and HCVRNA, and they are referred to as S₁, S₂, S₃, S₄, S₅ and S₆,respectively. Half of each liver specimen obtained from each of thepatients, respectively, were then fixed with formalin and subsequentlystained with HE and Azan to perform tissue diagnosis, while theremaining halves were subjected to grinding in a mortar with sea sand toobtain a supernatant. The supernatant was added into Eagle-MEM culturemedium (added with 10% FCS) wherein Chang cells are cultured, thenplaced in 95% air containing 5% CO₂ maintained at 37° C. to complete theinfection. The medium was then centrifuged at 1,800 rpm, and the cellsprecipitated were added into an Eagle-MEM culture medium (previouslyadded with FCS and antibiotic) containing UDCA (the concentration in themedium: 25 and 50 μM), one containing TUDCA (the concentration in themedium: 20 and 50 μM) and one without UDCA or TUDC respectively, andthen adjusted to the concentration of 1×10⁵ cells/slide. The cells werecultivated on LAB-TEK Chamber slides for one week in the UDCA-containingmedium and 2 weeks in the TUDCA-containing medium under 95% aircontaining 5% CO₂ maintained at 37° C.

The presence of HCV in these cultured Chang cells was firmly confirmedby the appearance of a band with 178 bp (base pair) by employing HCV RNAreverse transcription PCR method as described in Lancet Vol. 335, pages1419-22, 1990. Also, the infection rate of HCV to the Chang cells wasdetermined by immunologically staining HCV by using core antibody(Okayama's monoclonal antibody) and by counting the positive cells underan optical microscope. For the immunological staining, the ABC methodusing HRS was employed, wherein HRS is the abbreviation of Horse Radishstaining, while ABC is the abbreviation of Avidin-Biotin Complex. Inaddition, the infection of HCV to the Chang cells was also confirmedaccording to in situ hybridization method, separately.

The results of the inhibition test on the proliferation of HCV with UDCAand TUDCA are shown in Tables 1 and 2, respectively. The control in thetables shows the result obtained by subjecting to the same procedureexcept without the addition of the supernatant.

                  TABLE 1                                                         ______________________________________                                        Result of Inhibition Test on Proliferation of HCV with UDCA                   (Infection Rate: %)                                                                  Concentration of UDCA                                                         0 μM     25 μM 50 μM                                          ______________________________________                                        Control  29.7 ± 5.5 21.4 ± 0.9                                                                          22.2 ± 4.1                                 Patient S.sub.1                                                                        45.2 ± 4.2 35.6 ± 8.1                                                                          16.6 ± 2.0                                 Patient S.sub.2                                                                        50.1 ± 7.4 23.9 ± 7.0                                                                          17.2 ± 3.3                                 Patient S.sub.3                                                                        38.9 ± 8.2 27.0 ± 6.2                                                                          18.5 ± 2.8                                 ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Result of Inhibition Test on Proliferation of HCV with TUDCA                  (Infection Rate: %)                                                                  Concentration of TUDCA                                                        0 μM   20 μM  30 μM                                           ______________________________________                                        Control   8.4 ± 3.6                                                                             5.5 ± 5.0                                                                            2.8 ± 0.6                                   Patient S.sub.4                                                                        38.8 ± 7.1                                                                             32.8 ± 22.4                                                                          13.5 ± 8.1                                  Patient S.sub.5                                                                        18.2 ± 3.3                                                                             6.9 ± 3.4                                                                            6.8 ± 0.7                                   Patient S.sub.6                                                                        25.6 ± 7.3                                                                             9.3 ± 3.8                                                                            5.5 ± 1.2                                   ______________________________________                                    

As can be clearly seen in the Tables 1 and 2 shown above, theproliferation of HCV was significantly inhibited in both groupscontaining UDCA and TUDCA, respectively, in comparison with the groupnot containing UDCA or TUDCA.

(Clinical Test)

Each of 8 patients aged from 52 to 72 suffering from chronic hepatitisC, who are referred to S₇, S₈, S₉, S₁₀, S₁₁, S₁₂, S₁₃ and S₁₄,respectively, was orally administered with UDCA at a dose of from 1,200to 1,500 mg/day for a period of from 0.5 to 6 months. The quantitativevalues of HCV RNA in the serum of each of the patients before and afterthe dosage with UDGA were shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Quantitative Value of HCV RNA in Serum before and after Dosage with           UDCA                                                                                          Dosage  Quantitative Value of                                         Dose    Period  HCV RNA in Serum                                              (mg/day)                                                                              (Month) Before Dosage                                                                            After Dosage                               ______________________________________                                        Patient S.sub.7                                                                         1500      1       10.sup.9 /ml                                                                           10.sup.7 /ml                             (Female, Age 58)                                                              Patient S.sub.8                                                                         1500      1       10.sup.3 /ml                                                                           0/ml                                     (Male, Age 65)                                                                Patient S.sub.9                                                                         1200-1500 6       10.sup.10 /ml                                                                          10.sup.6.5 /ml                           (Male, Age 59)                                                                Patient S.sub.10                                                                        1200-1500 5       10.sup.10 /ml                                                                          10.sup.6 /ml                             (Female, Age 72)                                                              Patient S.sub.11                                                                        1500      1.5     10.sup.10 /ml                                                                          10.sup.6 /ml                             (Female, Age 60)                                                              Patient S.sub.12                                                                        1500      2       10.sup.10 /ml                                                                          10.sup.6.5 /ml                           (Female, Age 67)                                                              Patient S.sub.13                                                                        1500      0.5     10.sup.4 /ml                                                                           0/ml                                     (Male, Age 57)                                                                Patient S.sub.14                                                                        1200-1500 3       10.sup.4 /ml                                                                           0/ml                                     (Male, Age 52)                                                                ______________________________________                                    

As can be clearly seen from the Table 3 shown above, the proliferationof HCV in the patients suffering chronic hepatitis C was remarkablyinhibited by dosage with UDCA.

(Acute Toxicological Test)

Acute toxicological tests were carried out by using male and femaleWistar-strain rats aged 9 weeks and male and female dd-strain mice aged8 weeks for UDCA, and by using male and female Beagle dogs aged from 8to 11 months and male and female SD-strain rats aged 6 weeks for TUDCAto obtain LD₅₀, respectively. The results are shown in Tables 4 and 5,respectively.

                  TABLE 4                                                         ______________________________________                                        Result of Acute Toxicological Test on UDCA  LD.sub.50 (mg/Kg)!                           Rats            Mice                                                          Male  Female    Male    Female                                     ______________________________________                                        UDCA  Oral       >5000   >5000   >10000                                                                              >10000                                       Intravenous                                                                               310     320      285   240                                  ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Result of Acute Toxicological Test on TUDCA  LD.sub.50 (mg/Kg)!                          Beagle Dogs Rats                                                              Male   Female   Male     Female                                    ______________________________________                                        TUDCA  Oral      >5000    >5000  >10000 >10000                                       Intravenous                                                                             300-600  300-600                                                                              600-800                                                                              600-800                               ______________________________________                                    

As can be clearly seen from the Tables 4 and 5 shown above, both acutetoxicity of UDCA and TUDCA are very low, proving that these can be asafe drug.

UTILIZATION IN THE INDUSTRY

It was demonstrated that bile acid can remarkably inhibit theproliferation of hepatitis C virus and is less toxic, and therefore,bile acid can be useful as a hepatitis C virus proliferation inhibitor.

What is claimed is:
 1. A method of inhibiting hepatitis C proliferationin a mammal infected with hepatitis C virus, comprising administering tothe mammal a dosage consisting essentially of tauroursodeoxycholic acidor a physiologically acceptable salt thereof effective to inhibithepatitis C proliferation.
 2. A method according to claim 1, whereinsaid step of administering comprises oral administration in a dose rangeof 800 to 3,000 mg/day.
 3. A method of inhibiting hepatitis Cproliferation in a mammal infected with hepatitis C virus, comprisingadministering to the mammal a dosage consisting essentially ofchenodeoxycholic acid or a physiologically acceptable salt thereofeffective to inhibit hepatitis C proliferation.
 4. A method according toclaim 3, wherein said step of administering comprises oraladministration in a dose range of 800 to 3,000 mg/day.
 5. A method ofinhibiting hepatitis C proliferation in a mammal infected with hepatitisC virus, comprising administering to the mammal a dosage of bile acid ora physiologically acceptable salt thereof effective to inhibit hepatitisC proliferation, wherein said step of administering comprises oraladministration in a dose range of 800 to 3,000 mg/day.
 6. A methodaccording to claim 5, wherein said dose range comprises 1,200 to 1,800mg/day.
 7. A method according to claim 5, wherein said dose rangecomprises 1,400 to 1,600 mg/day.